Plastids are a family of closely related organelles that in one form or another are present in all living plant cells. All plastids share certain features. For example, they have their own small genome and are enclosed by an envelope composed of a double membrane. All plastids develop from protoplastids, relatively small organelles present in meristematic cells. Plastids develop according to the requirements of each differentiated cell. For instance, if the leaf is grown in darkness, the protoplastids develop into etioplasts that contain a yellow chlorophyll precursor called protochlorophyll. If, on the other hand, the leaves are grown in light, the etioplasts further develop into chloroplasts by converting protochlorophyll to chlorophyll. Chloroplasts are the site of photosynthesis, the process by which plants manufacture their own organic nutrients. Other forms of plastids are chromoplasts that accumulate carotenoid pigments. These plastids are responsible for the yellow-orange-red coloration of petals and fruit in many species. Leucoplasts are basically enlarged proplastids. They occur in many epidermal and internal tissues that do not become green and photosynthetic. Amyloplasts are a common form of leucoplasts. They store starch in storage tissues and, in certain cells of the stems, leaves and roots function as part of the plant response to gravity. All plastids contain multiple copies of the plastid genome and most are capable of division within the cell. The only type of cell and higher plant that loses its population of plastids is the male sperm cell in certain species. Thus, plastids of plants such as maize are maternally inherited. That is, they acquire their plastids solely from the egg cell. See, Alberts, et al., Molecular Biology of the Cell, Garland Publishing (New York), 1983, pp. 1120–1122.
The plastid genome of higher plants is a circular double-stranded DNA molecule of 120–165 kilobases that may be present in 2,000–50,000 copies per leaf cell. The plastid genome has become a very attractive target for genetic manipulation compared to the nuclear genome of the plant for several reasons. Since proteins in plastids may be expressed at a very high level, the molecular machinery of plastid is essentially a bacterial one. Also, a higher degree of containment can be achieved (no transmission via pollen) and because integration of heterologous DNA occurs via homologous recombination mechanism. DNA integrates randomly into the nuclear genome of a plant. The location of integration in the plastid genome, on the other hand, may be controlled such as by way of specific flanking sequences. There is no gene silencing or so-called position effects, so the level of expression is much more predictable. The level of expression is also much higher because there are many more DNA copies per plant cell. The chloroplast is basically a bacterium so it accommodates bacterial nucleic acid more readily than genomic DNA, without as much need for modification. This advantage applies equally to associated regulatory sequences such as bacterial promoters. The risk of gene release into the environment (referred to as “outcrossing”) is essentially eliminated because chloroplasts do not move into pollen. Lastly, since the chloroplast is the site of most important biosynthetic pathways e.g., starch, amino acids and fats, it is relatively easy to insert genes and have them function in the organelle of interest without a need for special targeting sequences.
Plastid transformation has proven extremely difficult, particularly in agronomically valuable crops. Most transformation methods are species- and variety-specific. Collectively, these limitations appear to reflect complex and uniquely species-specific ways in which transformed plastids are being selected in in vitro grown plant tissues. Only the reproducible production of transplastomic, fertile tobacco plants has been reported (Svab, et al., Proc. Natl. Acad. Sci. USA 87:8526–8530 (1990). A transplastomic Arabidopsis plant is reported in Sikdar, et al., Plant Cell Rep. 18:20–24 (1998) but these plants were sterile. The lack of success in this area despite the large investment being made illustrates the magnitude of the problem. Hence, there is a pressing need for methods of producing transplastomic plants.